The steps involved in the transcription and translation (expression) of genes in cells are complex but the basic steps for protein to be produced from DNA are transcription and translation. The DNA is first transcribed into RNA, and then the RNA is translated by the interaction of various cellular components into protein. In prokaryotic cells transcription and translation are coupled, meaning that RNA is translated into protein during the time that it is being transcribed from the DNA. In eukaryotic cells the two activities are separate, making the overall process more complicated. DNA is transcribed into RNA inside the nucleus of the cell, but the RNA is further processed into mRNA and then transported outside the nucleus to the cytoplasm where it is translated into protein.
The ability of molecular biologists to isolate and clone genes has brought about the development of systems that can be used to express the proteins encoded by these genes or their corresponding mRNA messages. Methods for expressing proteins make it possible to manipulate genes and then study the effect of the manipulations on their function. The amount of protein to be produced, whether the gene is prokaryotic or eukaryotic and the relative merits of an in vitro cell-free or an in vitro whole-cell system, are some of the factors considered by researchers when selecting an expression system.
In vitro transcription systems using prokaryotic or eukaryotic cells are available; however, these systems are difficult to work with since intact cells are used. In vitro cell-free systems, on the other hand, are made from cell-free extracts produced from prokaryotic or eukaryotic cells that contain all the necessary components to translate DNA or RNA into protein. Cell-free extracts can be prepared from prokaryotic cells such as E. coli and from eukaryotic cells such as rabbit reticulocytes and wheat germ. Cell-free systems are very popular because there are standard protocols available for their preparation and because they are commercially available from a number of sources. Although in vitro systems have many advantages to major draw back is that the amounts of protein produced is generally low which makes the analysis of protein function and activity difficult. The problems with low levels of protein production have forced researchers to develop complex assay systems using gel based systems and radioactivity that limit the application of these in vitro methods for studies of protein function and activity.
E. coli S30 cell-free extracts were first described by Zubay, G. (1973 Ann. Rev. Genet. Vol 7, p. 267). These extracts can be used when the gene to be expressed has been cloned into a vector containing the appropriate prokaryotic regulatory sequences, such as a promoter and ribosome-binding site. Prokaryotic E. coli cell-free systems are considered coupled because transcription and translation occur simultaneously after the addition of DNA to the extract.
Rabbit reticulocyte lysate was described by Pelham, H. R. B. and Jackson, R. J. (1976, Eur. J. Biochem. Vol. 67, p. 247). This expression system is probably the most widely used cell-free system for in vitro translation, and is used in the identification of mRNA species, the characterization of their products and the investigation of transcriptional and translational control.
Wheat germ extract was described by Roberts, B. E. and Paterson, B. M. (1973, Proc. Natl. Acad. Sci. U.S.A., Vol. 70, P. 2330). Cell-free extracts of wheat germ support the translation in vitro of a wide variety of viral and other prokaryotic RNAs, as well as eukaryotic mRNAs. (Anderson, C., et al. (1983) Meth. Enzymol. 101, 635). Generally, it is found necessary to include a ribonuclease inhibitor in the reaction mix of a wheat germ translation system, as ribonuclease activities in wheat germ extract are present.
Post-translational modifications that have been observed in rabbit reticulocyte lysate or wheat germ extract include signal peptide cleavage, glycosylation, acetylation, isoprenylation, proteolysis myristoylation, protein folding and proteolytic processing (Glass, C. A. and Pollard, K. M. (1990). Promega Notes 26). Some modifications or processing events have required the introduction of additional biological machinery. For example, processing events, such as signal peptide cleavage and core glycosylation, are examined by adding canine microsomal membranes (Walter, P. and Blobel, G. (1983) Meth. Enzymol. 96, 84) (Walter, P. and Blobel, G. (1983) Meth. Enzymol. 96, 84) or Xenopus egg extracts (Zhou, X, et al. U.S. Pat. No. 6,103,489) to a standard translation reaction. The addition of membrane preps is valuable when investigating membrane bound proteins such as receptors. These in vitro systems have been used to express many proteins, often in their native conformation and containing many of the normal and expected post-translational modifications.
RNA for translational studies can be obtained by either isolating mRNA or by making in vitro RNA transcripts from DNA that has been cloned into a vector containing an RNA polymerase promoter. The first method isolates mRNA directly from cells. The second obtains RNA for in vitro translation by in vitro transcription. In vitro transcription of cloned DNA behind phage polymerase promoters was described by Krieg, P. and Melton, D (1984, Nucl. Acids Res., Vol. 12, p. 7057). This method has become a standard method for obtaining RNA from cloned genes for use in in vitro translation reactions. The method uses DNA or a gene of interest that is cloned into a vector containing a promoter for an RNA polymerase. The vector is then purified and followed by an in vitro transcription reaction to make RNA transcripts. A number of vectors containing the SP6, T7 and T3 RNA polymerase promoters are commercially available and are widely used for cloning DNA.
After rabbit reticulocyte lysate and wheat germ extract were developed as cell-free translation systems, coupling of transcription and translation was demonstrated. One system that was developed was a linked transcription and translation system (Roberts, B. E., et al. (1975), Proc. Natl. Acad. Sci. U.S.A., Vol 72, 1922–1926). This system involved the use of wheat germ extract supplemented with E coli RNA polymerase and looked at transcription and translation of SV40 viral DNA. Another system was developed by Pelham, H. R. B, et al. (1978), Eur. J. Biochem., Vol. 82, 199–209, where coupled transcription and translation occurred after the introduction of vaccinia vital core particles into rabbit reticulocyte lysate.
Work has also been described using continuous cell-free in vitro translation systems with the emphasis on large-scale production of protein. Continuous translation involves a bioreactor (such as an Amicon 8MC ultrafiltration unit) in which large scale reactions are set up and protein is continually translated over extended periods of time. The reaction requires that a buffer be fed into the reaction as it progresses, and also requires that the products of translation be removed from the reaction filter unit. This type of system works well with E. coli S30 extract and wheat germ extract when RNA template is introduced. See Spirin, et al. (1988) Science, Vol 242, 1162–1164. The system also works using RNA templates in rabbit reticulocyte lysate. See Ryabova, et al. (1989) Nucl. Acid Res., Vol. 17, No. 11, 4412. The system is also known to work well with DNA templates in E. coli S30 extracts. See Baranov, et al. (1989) Gene, Vol 84, 463–466. PCT publication WO9102076 discloses continuous cell-free translation from DNA templates using eukaryotic lysates.
These methods of coupling transcription and translation have been further modified by coupling these in vitro transcription and in vitro translation steps in a single reaction mix from plasmid DNA containing RNA polymerase sites for the SP6, T7 or T3 RNA polymerases (Craig, D., et al (1992) Nucleic Acids Res. Vol 20, 4987–4995, U.S. Pat. No. 5,665,563; U.S. Pat. No. 5,324,637; U.S. Pat. No. 5,492,817; EP566 714). These coupled reaction mixes are available commercially from Promega both for the rabbit reticulocyte and wheat germ lysates.
The proteins from these translation or transcription translation reactions have been labeled using a variety of methods. This step is important given the fact that very small amounts of protein are produced in these translation reactions relative to the total amount of protein (4–0.4 ug/ml relative to 50–60 mg/ml of endogenous proteins).
One labeling method is the genetic modification of the gene or genes of interest, which results in the translation of a protein containing amino acids, amino acid sequences or post-translational modifications not found in the normal translation product of the gene. These modifications provide for the use of various methods for detection and purification of the translated proteins. These methods for the modification of the normal gene product are not always helpful as they can prevent the normal function of the protein due to the modification of the N terminal or C terminal portions of the protein. Also these modifications can prove to be costly and time consuming when applied on large scale.
Another labeling method involves the use of radioactive amino acids in translation reaction resulting in the production of a labeled amino acid sequence that is encoded by the DNA or RNA used to drive the translation reaction. Radioactive labeled amino acid sequences produced this way have been valuable in the study, detection and determination of these protein function and properties. The problems that are associated with the use of radioactivity are well known and make its use on a large scale problematic, unsafe and costly.
An alternative in vitro approach uses specific tRNAs linked to modified amino acids to produce amino acid sequences incorporating these modifications (Ohtsuka H, et al., Nucleic Acids Symp Ser. 1997;(37):125–6. Hoeltke H J, et al., Biotechniques. 1995;18(5):900–4, 906–7. Kurzchalia T V, et al. Eur J Biochem. 1988;172(3):663–8. McIntosh B, et al., Biochimie. 2000;82(2):167–74. Gite S, et al., Anal Biochem. 2000;279(2):218–25. Janiak F, et al., Biochemistry. 1990 8; 29(18):4268–77.). The best example of this approach is with the use of a Lys tRNA that is modified to contain a biotin. The resulting biotinyl-Lys-tRNA is used in translation reactions resulting in the production of proteins labeled with Biotin at random within the protein sequence. This method has been used for the detection of proteins on gels and for the purification of proteins prior to gel based analysis. This method for labeling proteins in combination with gel based detection methods has not provided a solution for rapid or simple assays for protein activity or function. Indeed some groups have found that this labeling method would not work when used in a solid phase assay due to endogenous biotinylase activity and high levels of endogenous lysine which markedly inhibit the ability to incorporate significant amounts of biotinylated lysine into newly synthesized proteins (Gao, Z-H., (2000) Biochem Biophys Res Comm, 268, 562–566).
Thus these methods for producing proteins and labeling them to date do not allow for the facile analysis of protein function or activity.
In order to improve on this method for labeling proteins other derivatives of tRNA have been developed. One example is the lysine tRNA labeled with the fluorophore, BODIPY-FL (Promega, Madison Wis. (Promega Notes 77)). This has improved the detection methodologies but is still based on using gel electrophoresis and in gel detection systems that are expensive, slow and complex.
At this time, there are a number of commercially available instruments that utilize electrochemiluminescence (ECL) for analytical measurements including drug screening. Species that can be induced to emit ECL (ECL-active species) have been used as ECL labels. Examples of ECL labels include: i) organometallic compounds where the metal is from, for example, the noble metals of group VIII, including Ru-containing and Os-containing organometallic compounds such as the tris-bipyridyl-ruthenium (RuBpy) moiety and ii) luminol and related compounds. Species that participate with the ECL label in the ECL process are referred to herein as ECL coreactants. Commonly used coreactants include tertiary amines (e.g., see U.S. Pat. No. 5,846,485), oxalate, and persulfate for ECL from RuBpy and hydrogen peroxide for ECL from luminol (see, e.g., U.S. Pat. No. 5,240,863. The light generated by ECL labels can be used as a reporter signal in diagnostic procedures (Bard et al., U.S. Pat. No. 5,238,808). For instance, an ECL label can be covalently coupled to a binding agent such as an antibody, nucleic acid probe, receptor or ligand; the participation of the binding reagent in a binding interaction can be monitored by measuring ECL emitted from the ECL label. Alternatively, the ECL signal from an ECL-active compound may be indicative of the chemical environment (see, e.g., U.S. Pat. No. 5,641,623 which describes ECL assays that monitor the formation or destruction of ECL coreactants). For more background on ECL, ECL labels, ECL assays and instrumentation for conducting ECL assays see U.S. Pat. Nos. 5,093,268; 5,147,806; 5,324,457; 5,591,581; 5,597,910; 5,641,623; 5,643,713; 5,679,519; 5,705,402; 5,846,485; 5,866,434; 5,786,141; 5,731,147; 6,066,448; 6,136,268; 5,776,672; 5,308,754; 5,240,863; 6,207,369 and 5,589,136 and Published PCT Nos. WO99/63347; WO00/03233; WO99/58962; WO99/32662; WO99/14599; WO98/12539; WO97/36931 and WO98/57154.
Commercially available ECL instruments have demonstrated exceptional performance. They have become widely used for reasons including their excellent sensitivity, dynamic range, precision, and tolerance of complex sample matrices. The commercially available instrumentation uses flow cell-based designs with permanent reusable flow cells. Recently, ECL instrumentation has been disclosed that uses reagents immobilized on the electrode used to induce ECL (see, e.g., U.S. Pat. No. 6,207,369 and Published PCT Application No. WO98/12539). Multi-well plates having integrated electrodes suitable for such ECL measurements have also been recently disclosed (see, e.g., copending Provisional Application No. 60/301,932 entitled “Assay Plates, Reader Systems and Methods for Luminescence Test Measurements”, filed on Jun. 29, 2001, and U.S. application Ser. Nos. 10/185,274 and 10/185,363, filed Jun. 28, 2002, each hereby incorporated by reference, each hereby incorporated by reference.
The use of multi-well assay plates allows for the parallel processing and analysis of multiple samples distributed in multiple wells of a plate. Typically, samples and reagents are stored, processed and/or analyzed in multi-well assay plates (also known as microplates or microtiter plates). Multi-well assay plates can take a variety of forms, sizes and shapes. For convenience, some standards have appeared for some instrumentation used to process samples for high throughput assays. Assays carried out in standardized plate formats can take advantage of readily available equipment for storing and moving these plates as well as readily available equipment for rapidly dispensing liquids in and out of the plates. Some well established multi-well plate formats include those found on 96-well plates (12×8 array of wells), 384-well plates (24×16 array of wells) and 1536-well plate (48×32 array of well). The Society for Biomolecular Screening has published recommended microplate specifications for a variety of plate formats (see, http://www.sbsonline.org), the recommended specifications hereby incorporated by reference.